Lipases
The lipases comprise a family of enzymes with the capacity to catalyze hydrolysis of compounds including phospholipids, mono-, di-, and triglycerides, and acyl-coa thioesters. Lipases play important roles in lipid digestion and metabolism. Different lipases are distinguished by their substrate specificity, tissue distribution and subcellular localization.
Lipases have an important role in digestion. Triglycerides make up the predominant type of lipid in the human diet. Prior to absorption in the small intestine, triglycerides are broken down to monoglycerides and free fatty acids to allow solubilization and emulsification before micelle formation in conjunction with bile acids and phospholipids secreted by the liver. Secreted lipases that act within the lumen include lingual, gastric and pancreatic lipases, each having the ability to act under appropriate pH conditions. Modulating the activity of these enzymes has the potential to alter the processing and absorption of dietary fats. This may be important in the treatment of obesity or malabsorption syndromes such as those that occur in the presence of pancreatic insufficiency.
Lipases have an important role in lipid transport and lipoprotein metabolism. Subsequent to absorption across the intestinal mucosa, fatty acids are transported in complexes with cholesterol and protein molecules termed apoliporoteins. These complexes include particles known as chylomicrons, very low density lipoproteins (“VLDLs”), low density lipoproteins (“LDLs”) and high density lipoproteins (“HDLs”) depending upon their particular forms. Lipoprotein lipase and hepatic lipase are bound to act at the endothelial surfaces of extrahepatic and hepatic tissues, respectively. Deficiencies of these enzymes are associated with pathological levels of circulating lipoprotein particles. Lipoprotein lipase functions as a homodimer and has the dual functions of triglyceride hydrolase and ligand/bridging factor for receptor-mediated lipoprotein uptake. Severe mutations that cause LPL deficiency result in type I hyperlipoproteinemia, while less extreme mutations in LPL are linked to many disorders of lipoprotein metabolism.
Lipases have an important role in lipolysis. Free fatty acids derived from adipose tissue triglycerides are the most important fuel in mammals, providing more than half the caloric needs during fasting. The enzyme hormone-sensitive lipase plays a vital role in the mobilization of free fatty acids from adipose tissue by controlling the rate of lipolysis of stored triglycerides. Hormone sensitive lipase is activated by catecholamines through cyclic AMP-mediated phosphorylation of serine-563. Dephosphorylation is induced by insulin. While mice with homozygous-null mutations of their hormone-sensitive lipase genes induced by homologous recombination have been shown to enlarged adipocytes in their brown adipose tissue and to a lesser extent their white adipose tissue, they are not obese. White adipose tissue from homozygous null mice retain 40% of their wild type triacylglycerol lipase activity suggesting that one or more other, as yet uncharacterized, enzymes also mediate the hydrolysis of triglycerides stored in adipocytes. Hormone-sensitive lipase does not show sequence homology to the other characterized mammalian lipase proteins.
The present invention has substantial similarity to lysosomal acid lipase. Human lysosomal acid lipase/cholesteryl ester hydrolase (EC 3.1.1.13) reveals that it is structurally related to enteric acid lipases, but lacks significant homology with any characterized neutral lipases.
The lysosomal enzyme catalyzes the deacylation of triacylglyceryl and cholesteryl ester core lipids of endocytosed low density lipoproteins; this activity is deficient in patients with Wolman disease and cholesteryl ester storage disease.
Its amino acid sequence, as deduced from the 2.6-kilobase cDNA nucleotide sequence, is 58 and 57% identical to those of human gastric lipase and rat lingual lipase, respectively, both of which are involved in the preduodenal breakdown of ingested triglycerides. Notable differences in the primary structure of the lysosomal lipase that may account for discrete catalytic and transport properties include the presence of 3 new cysteine residues, in addition to the 3 that are conserved in this lipase gene family, and of two additional potential N-linked glycosylation sites.
Two major disorders, the severe infantile-onset Wolman disease and the milder late-onset cholesteryl ester storage disease (CESD), are seemingly caused by mutations in different parts of the lysosomal acid lipase (LIPA) gene.
Burton and Reed (1981) demonstrated material crossreacting with antibodies to acid lipase in fibroblasts of 3 patients with Wolman disease and 3 with cholesterol ester storage disease. Quantitation of the CRM showed normal levels in both cell types. Enzyme activity was reduced about 200-fold in Wolman disease fibroblasts and 50- to 100-fold in cholesterol ester storage disease cells. The allelic nature of Wolman and cholesteryl ester storage diseases is the occurrence of possible genetic compounds, i.e., cases of intermediate severity (Schmitz and Assmann, 1989). In both Wolman disease and cholesteryl ester storage disease, Chatterjee et al. (1986) demonstrated that renal tubular cells shed in the urine are laden with cholesteryl esters and triacylglycerol and that LIPA is lacking in these cells. Yoshida and Kuriyama (1990) described lysosomal acid lipase deficiency in rats.
Aslanidis et al. (1994) summarized the exon structure of the LIPA gene, which consists of 10 exons, together with the sizes of genomic EcoRI and SacI fragments hybridizing to each exon. The DNA sequence of the putative promoter region was presented. Anderson et al. (1994) isolated and sequenced the gene for LIPA. They found that it is spread over 36 kb of genomic DNA. The 5-prime flanking region is GC-rich and has characteristics of a ‘housekeeping’ gene promoter.
Du et al. (1998) produced a mouse model of lysosomal acid lipase deficiency by a null mutation produced by targeting disruption of the mouse gene. Homozygous knockout mice produced no Lip1 mRNA, protein, or enzyme activity. The homozygous deficient mice were born in mendelian ratios, were normal appearing at birth, and followed normal development into adulthood. However, massive accumulation of triglycerides and cholesteryl esters occurred in several organs. By 21 days, the liver developed a yellow-orange color and was up to 2 times larger than normal. The accumulated cholesteryl esters and triglycerides were approximately 30-fold greater than normal. The heterozygous mice had approximately 50% of normal enzyme activity and did not show lipid accumulation. Male and female homozygous deficient mice were fertile and could be bred to produce progeny. This mouse model is the phenotypic model of human CESD and a biochemical and histopathologic mimic of human Wolman disease.
For a review related to lysosomal acid lipase, see Anderson et al., Proc. Nat. Acad. Sci. 91: 2718-2722, 1994; Anderson et al., Genomics 15: 245-247, 1993; Anderson et al., J. Biol. Chem. 266: 22479-22484, 1991; Aslanidis et al., Genomics 20: 329-331, 1994; Aslanidis et al., Genomics 33: 85-93, 1996; Assmann et al., In: Stanbury, J. B.; Wyngaarden, J. B.; Fredrickson, D. S.; Goldstein, J. L.; Brown, M. S. : Metabolic Basis of Inherited Disease. New York: McGraw-Hill (pub.) (5th ed.) 1983. Pp. 803-819; Beaudet et al., J. Pediat. 90: 910-914, 1977; Besley et al., Clin. Genet. 26: 195-203, 1984; Burton et al., Am. J. Hum. Genet. 33: 203-208, 1981; Byrd et al., Acta Neuropath. 45: 37-42, 1979; Cagle et al., Am. J. Med. Genet. 24: 711-722, 1986; Chatterjee et al., Clin. Genet. 29: 360-368, 1986; Christomanou et al., Hum. Genet. 57: 440-441, 1981; Coates et al., Am. J. Med. Genet. 2: 397-407, 1978; Crocker et al., Pediatrics 35: 627-640, 1965; Desai et al., Am. J. Med. Genet. 26: 689-698, 1987; Di Bisceglie et al., Hepatology 11: 764-772, 1990; Du et al., Hum. Molec. Genet. 7: 1347-1354, 1998; Fujiyama et al., Hum. Mutat. 8: 377-380, 1996; Hoeg et al., Am. J. Hum. Genet. 36: 1190-1203, 1984; Kahana et al., Pediatrics 42: 70-76, 1968; Klima et al., J. Clin. Invest. 92: 2713-2718, 1993; Koch et al., Somat. Cell Genet. 7: 345-358, 1981; Koch et al., Cell Genet. 25: 174, 1979; Konno et al., Tohoku J. Exp. Med. 90: 375-389, 1966; Lake et al., J. Clin. Path. 24: 617-620, 1971; Lake et al., J. Pediat. 76: 262-266, 1970; Lough et al., Arch. Path. 89: 103-110, 1970; Marshall et al., Arch. Dis. Child. 44: 331-341, 1969; Maslen et al., Am. J. Hum. Genet. 53 (suppl.): A926, 1993; Muntoni et al., Hum. Genet. 95: 491-494, 1995; Muntoni et al., Hum. Genet. 97: 265-267, 1996; Pagani et al., Hum. Molec. Genet. 5: 1611-1617, 1996; Patrick et al., Nature 222: 1067-1068, 1969; Roytta et al., Clin. Genet. 42: 1-7, 1992; Schaub et al., Europ. J. Pediat. 135: 45-53, 1980; Schiffet al., Clinical aspects. Am. J. Med. 44: 538-546, 1968; Schmitz et al., The Metabolic Basis of Inherited Disease. New York: McGraw-Hill (pub.) (6th ed.) 1989. Pp. 1623-1644; Sloan et al., J. Clin. Invest. 51: 1923-1926, 1972; Spiegel-Adolf et al., Confin. Neurol. 28: 399-406, 1966; Wolman et al., Pediatrics 28: 742-757, 1961; Yokoyama et al., J. Inherit. Metab. Dis. 15: 291-292, 1992; Yoshida et al., Lab. Animal Sci. 40: 486-489, 1990; Young et al., Arch. Dis. Child. 45: 664-668, 1970.
As identified above and in the cited references, lipase proteins are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of the lipase family of proteins. The present invention advances the state of the art by providing previously unidentified human proteins that have homology to known members of the lipase family of proteins.
Lipase proteins, particularly members of the lysosomal acid lipase subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of lipase proteins. The present invention advances the state of the art by providing a previously unidentified human lipase proteins that have homology to members of the lysosomal acid lipase subfamily.